Antisense Elements (Genetics)

Abstract: Intraperitoneal injection of an unmodified antisense peptide nucleic acid (PNA) complementary to mRNA of the rat neurotensin (NT) receptor (NTR1) was demonstrated by a gel shift assay to be present in brain, thus indicating that the PNA had in fact crossed the blood–brain barrier. An i.p. injection of this antisense PNA specifically inhibited the hypothermic and antinociceptive activities of NT microinjected into brain. These results were associated with a reduction in binding sites for NT both in brain and the small intestine. Additionally, the sense-NTR1 PNA, targeted to DNA, microinjected directly into the brain specifically reduced mRNA levels by 50% and caused a loss of response to NT. To demonstrate the specificity of changes in behavioral, binding, and mRNA studies, animals treated with NTR1 PNA were tested for behavioral responses to morphine and their mu receptor levels were determined. Both were found to be unaffected in these NTR1 PNA-treated animals. The effects of both the antisense and sense PNAs were completely reversible. This work provides evidence that any antisense strategy targeted to brain proteins can work through i.p. delivery by crossing the normal blood–brain barrier. Equally important was that an antigene strategy, the sense PNA, was shown in vivo to be a potentially effective therapeutic treatment.

Abstract: Up to now, out of approximately 20 antisense oligodeoxyribonucleotides (as ODN) selected and tested against a given target gene, only one species shows substantial suppression of target gene expression. In part, this seems to be related to the general assumption that the structures of local target sequences or antisense nucleic acids are unfavorable for efficient annealing. Experimental approaches to find effective as ODN are extremely expensive when including a large number of antisense species and when considering their moderate success. Here, we make use of a systematic alignment of computer-predicted secondary structures of local sequence stretches of the target RNA and of semi-empirical rules to identify favorable local target sequences and, hence, to design more effective as ODN. The intercellular adhesion molecule 1 (ICAM-1) gene was chosen as a target because it had been shown earlier to be sensitive to antisense-mediated gene suppression. By applying the protocol described here, 10 ICAM-1-directed as ODN species were found that showed substantially improved inhibition of target gene expression in the endothelial cell line ECV304 when compared with the most effective published as ODN. Further, 17 out of 34 antisense species (50%) selected on the theoretical basis described here showed significant (>50%) inhibition of ICAM-1 expression in mammalian cells.

Abstract: The ability of specific nucleotides to base-pair, or hybridise, is fundamental to the role of nucleic acids in information transfer. The elucidation of the complementary nature of the two strands of the DNA helix was soon followed by the in vitro demonstration of DNA: DNA hybridisation, RNA:DNA hybridisation, and RNA:RNA hybridisation. Initially hybridisation was employed analytically to monitor particular classes of RNA in cell extracts, or to deduce the presence of repetitive DNA sequences in the eucaryotic genome (see Lewin, 1974). However, in the late 1970s, for the first time, in vitro hybridisation was used to interfere with the translation of specific mRNAs by a process known as hybrid arrest of translation (Paterson et al. 1977). Subsequently, the availability of cloned genes and synthetic oligonucleotides has permitted many attempts to interfere with gene expression in vivo by exploiting the tendency of complementary sequences to hybridise. The principle of the antisense approach is to inhibit the expression of a specific gene by the provision of complementary RNA or DNA sequences that will hybridise to the transcripts of the gene. In this review I will assess the rationale behind antisense experiments, the antisense reagents and, finally, attempt a critical analysis of the successes and failures of this fashionable technique, of which it might be fairly said that it has promised more than it has delivered. I will confine most of my analysis to experiments that use antisense strategies to understand the contribution of a gene to cellular and developmental processes. I will not review the burgeoning applications of antisense reagents for therapeutic purposes in man or commercial purposes in plants. For reviews that incorporate these developments the reader is directed to Krol et al. (1988a), Cohen (1990) and Rossi and Sarver (1990).

Abstract: Since their invention in 1991, peptide nucleic acids (PNAs) have been used in a myriad of chemical and biological assays. More recently, peptide nucleic acids have also been demonstrated to hold great potential as therapeutic agents because of their physiological stability, affinity for target nucleic acids, and versatility. While recent modifications in their design have further improved their potency, their preclinical development has reached new heights due to their combination with recent advancements in drug delivery. This review focuses on recent advances in PNA therapeutic applications, in which chemical modifications are made to improve PNA function and nanoparticles are used to enhance PNA delivery.